Multimodal magnetic resonance imaging after experimental moderate and severe traumatic brain injury: A longitudinal correlative assessment of structural and cerebral blood flow changes

Traumatic brain injury (TBI) is a worldwide problem that results in death or disability for millions of people every year. Progressive neurological complications and long-term impairment can significantly disrupt quality of life. We demonstrated the feasibility of multiple magnetic resonance imaging (MRI) modalities to investigate and predict aberrant changes and progressive atrophy of gray and white matter tissue at several acute and chronic time points after moderate and severe parasagittal fluid percussion TBI. T2-weighted imaging, diffusion tensor imaging (DTI), and perfusion weighted imaging (PWI) were performed. Adult Sprague-Dawley rats were imaged sequentially on days 3, 14, and 1, 4, 6, 8, and 12 months following surgery. TBI caused dynamic white and gray matter alterations with significant differences in DTI values and injury-induced alterations in cerebral blood flow (CBF) as measured by PWI. Regional abnormalities after TBI were observed in T2-weighted images that showed hyperintense cortical lesions and significant cerebral atrophy in these hyperintense areas 1 year after TBI. Temporal DTI values indicated significant injury-induced changes in anisotropy in major white matter tracts, the corpus callosum and external capsule, and in gray matter, the hippocampus and cortex, at both early and chronic time points. These alterations were primarily injury-severity dependent with severe TBI exhibiting a greater degree of change relative to uninjured controls. PWI evaluating CBF revealed sustained global reductions in the cortex and in the hippocampus at most time points in an injury-independent manner. We next sought to investigate prognostic correlations across MRI metrics, timepoints, and cerebral pathology, and found that diffusion abnormalities and reductions in CBF significantly correlated with specific vulnerable structures at multiple time points, as well as with the degree of cerebral atrophy observed 1 year after TBI. This study further supports using DTI and PWI as a means of prognostic imaging for progressive structural changes after TBI and emphasizes the progressive nature of TBI damage.

Introduction Traumatic brain injury (TBI) is a critical global health problem that results in death or disability for millions of people every year [1]. TBI is a causal factor in 34 percent of all injury-related deaths in the United States [2]. In addition, The Center for Disease Control and Prevention [3] reported that 2.8 million individuals sustain a TBI each year. Falls, motor vehicle collisions, sports-related injuries, abuse/assault, and blast-induced high-pressure waves are the primary causes of TBI [2].
TBI can result in an extensive range of deleterious consequences, from cognitive deficits and disabling neuropsychiatric symptoms to coma and death [4][5][6]. Unfortunately, the full extent of symptomatology is often not fully realized until several years after the injury due to the progressively degenerative nature of TBI and evolving histopathological changes [7]. Additionally, TBI has been reported to be a risk factor for the development of Alzheimer's disease (AD) and other neurodegenerative disorders [4,[7][8][9][10].
Previous clinical and preclinical studies have reported progressive atrophy of specific gray and white matter structures after various types of TBI [8,11,12]. These progressive changes are associated with chronic deficits in a number of neurological disorders [13][14][15][16][17][18]. In clinical studies, anatomical injuries resulting from TBI include contusions in highly susceptible regions, such as the frontal and temporal lobes [19]. In addition, opposing pressure gradients generated by the primary mechanical injury can alter vulnerable tissue microarchitecture. Stretching, compression, and rotation of susceptible tissue often results in multi-focal white matter abnormalities and diffuse axonal injury (DAI)/traumatic axonal injury (TAI), leading to progressive alterations in axonal transport and function [20]. These phenomena occur hours to days after injury, possibly creating multiple therapeutic windows for treatment [11,[21][22][23]. Recent studies have shown that DAI is correlated with long-term consequences associated with neurodegenerative processes that can persist for years after the injury and may contribute to deleterious neurological events such as Alzheimer's disease-like pathological changes [10,24].
Neuroimaging is essential in diagnosing, classifying, and for the management of TBI patients. Focal contusions and lesions can be visualized using computed tomography (CT) scan and conventional magnetic resonance imaging (MRI), however the visualization of these acute injuries is often unable to predict clinical outcomes and is not always sufficiently sensitive to detect microscopic secondary changes after TBI [25][26][27][28][29]. One explanation for this gap in imaging and functional outcomes is that conventional imaging techniques miss the presence of DAI, and the disruption of white matter integrity and subsequent alterations in more remote subcortical regions are not fully appreciated with single imaging approaches, which contributes to the degree of deficit severity and correlates to overall functional outcomes. Observation of white matter hemorrhages in neuroimaging is interpreted as coincident with DAI, however 80% of axonal injuries are non-hemorrhagic [30]. Skandsen et al. reported DAI was observed in 72% of patients presenting with moderate to severe head injury [31].
Diffusion tensor imaging (DTI) is an advanced non-invasive MRI technique that provides critical information regarding white matter tract integrity by measuring water diffusion and changes in anisotropy [26,32,33]. Parametric maps, including fractional anisotropy (FA), apparent diffusion coefficient (ADC), and radial diffusivity (RD), allow for the determination of the directional flow and permissibility of water molecule diffusion through tissue and hence the subsequent integrity or compromise of white matter [34].
ADC reflects the diffusion speed of water molecules and can be utilized to determine the degree of obstruction of water molecule diffusion in turn providing information regarding histological architecture [35]. In DTI, changes in FA can be reflective of alterations of several elements, such as axonal membrane integrity, myelination, axon diameter, and axon attenuation [36]. Alterations in FA are significantly correlated with cognitive deficits in moderate and severe TBI patients, and have been recognized as one of the most sensitive markers of TBIinduced cognitive and motor impairment [37]. RD is a scalar measurement of diffusion that is perpendicular to the length of an axon, and it, along with FA, are considered proxies for fiber organization and integrity of myelination. RD values are also highly sensitive to DAI and TAI [38].
Hemodynamics and cerebrovascular changes are observed after TBI as preclinical studies have reported impaired cerebral vascular autoregulation, microvascular injury, and increased blood-brain barrier permeability, resulting in changes of cerebral blood flow (CBF), ischemia, and infarction [39][40][41][42][43]. Kochanek and colleagues reported a significant reduction in cortical and hippocampal CBF 1 year after controlled cortical impact injury, which was concomitant with tissue loss in these structures [44]. Microvascular abnormalities at various posttraumatic time periods after TBI are associated with DAI and in subsequent cognitive and functional impairment [45,46]. Therefore, another valuable, non-invasive imaging modality is perfusionweighted imaging (PWI), which provides information on the hemodynamic state of the brain and allows for the detection of alterations in perfusion of the injured area and penumbral regions of the brain.
In the present study, we sought to investigate the ongoing and progressive nature of structural and hemodynamic consequences after moderate and severe TBI using an established model that produces a pressure-induced gliding contusion at the gray/white matter interface resulting in both focal and diffuse gray and white matter injury [47]. Through the longitudinal assessment of DTI and PWI alterations we investigated how those changes are reflected in chronic atrophic tissue at 12 months post injury. We propose that using both DTI and PWI modalities at several time points will provide an additional level of analysis and better illuminate the degree of neurological dysfunction after trauma and thus identify both early and later occurring cellular and physiological therapeutic targets for TBI.

Animals
Adult 3-to 4-month-old male Sprague Dawley rats (Rattus norvegicus; n = 26) were randomly assigned to 1 of 3 groups: uninjured sham (n = 10), moderate TBI (n = 8), or severe TBI (n = 8). Blocking randomization and standardization strategies were used to enhance scientific rigor and to control for variables that could potentially bias the results and ensure balanced numbers in each treatment group [48]. Females were not utilized in the present study as hormonal fluctuations associated with the female reproductive cycle may introduce biological variability. To standardize TBI models, monitoring and controlling for physiological and metabolic variables is important to reduce variability. In accordance with this, to determine the minimum number of animals needed for these studies and obtain meaningful data, a sample size analysis using SigmaPlot software 14.5 was performed for each outcome measure. Power was set to 0.80, minimum detectable difference in means, expected standard deviation of residuals, number of groups and alpha at 0.05 were calculated for each outcome to determine sample size. A sample size of n = 8-10 per group was obtained. All experimental procedures were compliant with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the University of Miami Institutional Animal Care and Use Committee, and in compliance with Animal Research Reporting In vivo Experiments (ARRIVE) guidelines. Animals were housed in a temperature-controlled room (22˚C) and exposed to a 12 h light/dark cycle. They were acclimated for at least 7 days prior to surgery.

Fluid-percussion injury
On Day 1 of surgery, animals were anesthetized with 3.0% halothane and a mixture of 70% nitrous oxide (N 2 O) and 30% oxygen (O 2 ). Once an adequate level of anesthesia was reached, the animals were placed in a stereotaxic frame. A 4.8 mm craniotomy was made overlying the right parietal cortex (3.8 mm posterior to bregma and 2.5 mm lateral to the midline), and a plastic injury tube (3.5 mm inner diameter) was adhered to the skull over the intact dura. The scalp was then closed, and animals were allowed to recover before being returned to their home cage. At this point, food was removed, and animals were fasted overnight to maintain consistent glucose levels, which can affect TBI outcomes [49,50].
Twenty-four hours later, animals were reanesthetized with 3.0% halothane in a mixture of 70% N 2 O and 30% O 2 . The right femoral artery was cannulated (PE-50 tubing), and blood samples were collected at regular intervals to monitor blood gases (pO 2 and pCO 2 ), blood pH, glucose, and mean arterial blood pressure (MABP). Rats were intubated, immobilized with pancuronium bromide (1.0mg/kg IA), mechanically ventilated, and maintained on 0.5-1.0% halothane in a mixture of 70% N 2 O, and 30% O 2 . For TBI groups, an FPI device was used to induce moderate (1.8-2.2 atmosphere) or severe (2.3-2.5 atmosphere) parasagittal FPI [40,51]. Physiological variables were maintained in normal ranges 15 min prior to TBI and for up to 30 min post-injury (pO 2 , 105-140 mmHg; pCO 2 , 35-45 mmHg; pH, 7.35-7.45), and brain and body temperatures were maintained at 37˚C using a feedback heat lamp system. Pain management measures were taken to alleviate discomfort. Buprenorphine was given once after surgery (0.01 mg/kg, subcutaneously). Sham animals underwent identical procedures minus the physical fluid percussion insult. Animals were allowed to survive for 1 year and were then sacrificed for histological analysis.

Physiology
All physiological variables, including pH, pO 2 , pCO 2 , and MABP were maintained within normal ranges before and after FPI. Animals showed normal activity within 24 h after recovery from anesthesia, which was determined by observation of grooming behavior, posture, and locomotion. There was no significant weight loss in any experimental group. No animals were excluded after surgery.

Magnetic resonance imaging
Magnetic resonance measurements were performed on each animal during critical early time points (Days 3 and 14) and at several chronic time points (1,4,6,8, and 12 months) post-surgery utilizing a 4.7-Tesla (200-MHz) 30-cm bore magnet interfaced to a Bruker Advance console. The investigators were blinded to the condition of the animal. During the MRI sessions, animals were anesthetized with 1.0% halothane in N 2 O/O 2 (70%/30%) and placed in an MRIcompatible cradle in the prone position utilizing a custom-built plexiglass holder with the head and nose securely fixed to minimize motion artifacts. Warm water (37 ± 0.5˚C) was circulated through the animal cradle to control the temperature of the animals. Volumetric measurements of the brain were performed by utilizing a high-resolution T 2 rapid acquisition relaxation-enhanced (RARE) multi-slice acquisition. Twenty-five slices were acquired to include all structures used in the histopathological analysis. The imaging parameters were as follows: a RARE factor of 8, TR4500 ms, effective TE 67.5 ms, field of view 3.00 cm, image matrix size of 184X184 (163 μm in-plane resolution) and slice thickness of 500 μm. Additionally, DTI datasets were acquired utilizing 8 independent diffusion directions, employing a bvalue of 0 and 1000s/mm 2 , a TR of 1500 ms, a TE of 29 ms, a matrix size of 128x128 (234 μm in-plane resolution) and a slice thickness of 1 mm. Lastly, continuous arterial spin labeling (cASL) EPI perfusion datasets were collected to calculate CBF maps of the brain. A 4-shot segmented EPI scan was used for the cASL experiments utilizing a TE of 50 ms, a TR of 1800 ms, a field of view of 3.0 cm, image matrix size of 128X128 (234 μm in-plane resolution) and slice thickness of 1.5 mm. The labeling settings employed for the cASL studies were a 1500 ms labeling period utilizing a 200 ms post-labeling delay and a 10mT/m labeling gradient. The total time for imaging was 1 h 30 min per session.

Volumetric analysis (T2 datasets)
Regions of interest (ROIs) were manually defined on the T2-weighted datasets on the Bruker Advance console by a blinded investigator [52]. A program was developed to read in polygan convex coordinates of the ROIs to export the reconstructed ROIs from the Bruker console to different computer platforms. Based on different scanned slice thickness (T2: 0.5 mm, DTI: 1 mm, PWI: 1.5 mm), we selected the T2 sections corresponding to the same anatomical sections of the DTI/PWI images and superimposed reconstructed ROIs on top of the DTI/PWI images to acquire ROI data in terms of mean and standard deviation.

Diffusion tensor analysis
DTI datasets were analyzed by a blinded investigator using DTI-Studio™ created by Susumu Mori et al. at Johns Hopkins University [53]. This software was employed to create ADC maps, FA maps, RD [34] maps and principle Eigen value maps (EVA0) maps. ADC and RD metrics are presented in mm 2 /s. FA is expressed in arbitrary units (a.u.). ROIs were first defined on the T2 weighted image obtained during the same imaging session and at the same neuroanatomical level as the diffusion-weighted images (DWI), and then transferred onto the DTI parameter maps.

Perfusion weighted imaging
PWI datasets were generated by the Bruker scanner. Based on the published method [54], using water as a freely diffusible tracer, we present the CBF images measured in unit of ml/g/ minute [46].

Quantitative histopathology
At 1-year post TBI or sham procedures, animals were reanesthetized with 3% halothane in 30% O 2 and 70% N 2 O, and transcardially perfused with saline (80 ml for 2 min) followed by 4% paraformaldehyde (4˚C, 350 ml at a pressure of 100-120 mm Hg for 28 min) by a surgeon blind to the condition of the animal. After decapitation, the brains were immediately removed and placed in 4% paraformaldehyde at 4˚C for 48 h. Brains were then blocked and embedded in paraffin for tissue sectioning. Coronal tissue sections were acquired at 10 μm thickness on a microtome (Leica HM 2125) at 150 μm intervals. One series of sections were stained with Luxol Fast Blue and hematoxylin-eosin (H&E) for volumetric assessment. Brain volume was measured by contouring the ipsilateral and contralateral cortex, corpus callosum (CC), external capsule (EC), and hippocampus at 7 specific bregma levels (-0.8, -1.8, -3.3, -4.3, -5.8, -6.8-, and -7.3-mm posterior to bregma) [55]. Volumes were determined by tracing the boundaries of each structure at a power of 10x from a minimum of two coronal sections at each of the bregma levels, using a camera lucida microscope attachment. To determine the differences in tissue shrinkage, percent atrophy was determined by calculating the difference between ipsilateral and contralateral volume and normalizing to the contralateral volume. Representative images were acquired at 20x using an Olympus BX51 microscope (Olympus America), and the volume estimation was carried out using Neurolucida software (version 7.50.1, Micro Brightfield Inc., Willinston, ME) by a blinded investigator.

Statistical analysis
All statistical analysis procedures were blindly conducted on GraphPad Prism (v 9.5.0). Data are expressed as mean ± standard error of the mean (SEM). Intergroup differences among bilateral structural volumes, DTI measurements, and PWI values were evaluated using one-or two-way repeated measures analysis of variance (ANOVA). In the case of missing data points, mixed-effects model analysis is conducted. The Shapiro-Wilk test (alpha = 0.05) was applied to determine if data were normally distributed and Bartlett's test to determine if standard deviations were significantly different. Post-hoc multiple comparison analysis was performed using Tukey corrections to control for the probability of making at least one Type 1 error across all pairwise comparisons. Significance level was p < 0.05. Exact p-values, Mean differences, SE of differences, q statistics, degrees of freedom (DF), 95% CI of ipsilateral differences are reported in the text and in entirety in S1 Table. Correlational analyses were carried out comparing all ADC, RD, and FA metrics to PWI CBF and histopathological indices within each structure at each imaging time point across groups. Correlations between parameters that were significant were determined using simple linear regression analysis and r Pearson values reported. Only significant correlations were reported. Full regression analysis results are reported in S2 Table.

Results
Vulnerable regions are affected in TBI, resulting in hallmark sequelae across patients. We investigated key ROIs susceptible to functional and structural damage after brain injury (Fig 1,  top). After undergoing moderate or severe fluid-percussion TBI or sham surgery, animals were imaged longitudinally 7 consecutive times using described multiple MRI modalities. Across time points, T2-weight images illustrated progressive anatomical pathology in both injury groups within the injured ipsilateral (right-hand side) hemisphere (Fig 1, bottom).

Diffusion tensor imaging
DTI metrics were assessed in the cortex, hippocampus, corpus callosum, and external capsule ROIs on the ipsilateral side where the injury was generated. Several quantitative measurements were obtained, including ADC, FA, and RD. In measuring ADC across ROIs and injury groups, we observed an unexpected dip in values at an acute 3-day time point, reaching significance between sham and moderately injured animals in the hippocampus and external capsule (p = 0.0134; DF 14.72 and p = 0.0332; DF 15.48, respectively) (Fig 2). In the ipsilateral hippocampus, ADC values acquired at 3 days post moderate TBI were significantly disparate from ADC values at virtually all subsequent imaged time points in this animal group. As time progressed, ADC increased and remained elevated throughout the imaging, with a high incidence of significance seen in the ipsilateral hippocampus and external capsule between both sham versus moderate and sham versus severe injury groups. At 8 months post TBI, severely injured animals had significantly elevated ADC values relative to moderately injured groups in the corpus callosum white matter tract (see S1 Table for all corresponding statistical measurements and exact p-values).
As mentioned previously, FA measures fiber directionality and can be interpreted as an indicator of axonal dysfunction [36,56]. In the present study, we did not observe any significant changes in the cortex at any time point compared to control, however there were significant differences between moderate, severe, and sham groups in all other ROIs, primarily in the ipsilateral external capsule (Fig 3). Furthermore, most of the significant FA reduction was evident at more chronic time points after injury, except for in the external capsule in which there were significant acute reductions among moderate and severe groups versus sham animals. The hippocampus, in which ADC alterations were highly significant across all time points, was only moderately affected in FA assessments with significant differences only observed between sham and severe FPI groups at two time points.
We also acquired RD metrics, a DTI assessment of the perpendicularity of fibers and of white matter functionality, to further evaluate microstructural alterations in white matter between uninjured and injured groups across time points. We observed that RD values resembled those of FA in that there were significant pathological elevations after both moderate and severe injury relative to sham controls in all ROIs except for the cortex (Fig 4). Analysis of this parameter showed the greatest significance in the hippocampus, followed by external capsule and corpus callosum in both subacute and chronic time points.
Taken together, our DTI assessments revealed that 1) ADC was significantly and bidirectionally altered in all ipsilateral ROIs evaluated between sham and injured groups; 2) FA values were

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significantly reduced in ipsilateral hippocampus and corpus callosum chronically, while reduced in the external capsule at both acute and chronic time points; 3) RD values were significantly aberrantly elevated in ipsilateral hippocampus and in major white matter tracts at the majority of time points except for 3 days post TBI; 4) DTI differences among groups were primarily observed between sham versus moderate and sham versus severe groups, however at 8 months post injury, all DTI acquisitions revealed significant differences between moderate TBI and severe TBI groups. We did not observe any significant differences in contralateral ROIs among groups.

CBF changes after moderate and severe TBI
In addition to DTI, CBF values were also acquired from all groups and across all time points using PWI (S1 Table). We detected a perceptible drop in CBF levels in the cortex across all  S1 Table for values and groups exhibiting significance). ADC in the corpus callosum was primarily statistically significant between sham and severe groups; however, at 8 months, significant differences were also observed between moderate and severe animals. The external capsule exhibited statistically different ADC at all, but a 4-month time point between both shams versus moderate and sham versus severe groups. *p � 0.05; **p � 0.01. https://doi.org/10.1371/journal.pone.0289786.g002

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time points in both injury groups, reaching significance among sham versus moderate and severe at 4 months post injury, and between sham and severe at 6 months (Fig 5). In the hippocampus, an acute 3 day decrease in blood flow was recorded between sham and moderately injured animals. This acute reduction was followed by signficant decreases in blood flow at more chronic time points out until 12 months. There were no statistically significant CBF differences between moderate and severe TBI groups at any time point nor within contralateral ROIs.

Chronic histopathology
At 12 months post moderate, severe, or sham surgery, all animals were sacrificed and assessed in order to detect the extent of cerebral atrophy. An asymmetry index was calculated based on structural volumes assessing atrophy between ipsilateral and contralateral sides. The greater FA in the cortex initially dropped and then increased by 1 month, however, these changes were not significantly different from sham. FA values in the hippocampus were aberrantly reduced after severe injury at chronic 8-and 12-month time points. Chronic FA differences were also evident in the corpus callosum, but only between sham and moderate groups. In the external capsule, FA was reduced at several acute and chronic time points between sham and injury groups and between moderate and severe groups at 8 months post-TBI. *p � 0.05; **p � 0.01; ***p� 0.001; ****p� 0.0001. https://doi.org/10.1371/journal.pone.0289786.g003

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value of the asymmetry index, the greater the degree of atrophic difference between the injured and uninjured hemispheres within each animal. Volumetric analyses of the posttraumatic atrophic cytoarchitecture revealed high levels of statistical significance between ipsilateral and contralateral ROIs in both moderate and severe injured animals relative to sham animals ( Fig  6). Twelve-month histological assessments showed no differences in asymmetry indices between moderate and severe animals.

Can DTI and PWI modalities be used as prognostic indicators?
Changes in the cerebral cortex. As shown in Fig 2, one month after severe TBI, ADC levels were aberrantly elevated in all structures, including within the cortex, consistent with Similar to what was observed in FA, RD values recorded in ipsilateral ROIs exhibited statistically significant increases compared to control groups except for the cortex. Within the hippocampus, RD was pathologically elevated at every time point between sham and injury groups except for at an acute phase 3 days post TBI. In the corpus callosum, there was statistically abnormal heightened RD in the majority of time points between sham and injured animals, while imaging at 8 months revealed a significant increase between moderate and severe RD values. The external capsule had elevated RD from 1 month until the 12-month endpoint between sham and TBI groups.

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expected pathological fluctuation patterns. Correlation analyses were conducted which revealed that lower ADC values were associated with greater CBF perfusion in cortical tissue at a one-month time point (Fig 7, top left). Additionally, as shown in Fig 5, we detected a significant difference in PWI between sham and severely injured animals at a 6-month time point. Using correlation analysis, we found that statistically-significant decreased CBF at 6 months negatively correlated with worsened histopathology (Fig 7, top right) indicative that reduced blood flow at 6 months was associated with increased cortical atrophy at 12 months after severe TBI.
Changes in the hippocampus. In severe groups, we found that at 6-and 8-month chronic time points, FA DTI values positively correlated with PWI CBF in ipsilateral hippocampal tissue (Fig 7, middle row). This indicates that at these times points, worsened FA outcomes in terms of reduction relative to sham controls correlated with worsened CBF perfusion in the hippocampus after severe TBI. DTI and CBF correlation was not observed in moderate animals.
Changes in the corpus callosum. In the corpus callosum, we observed that acute ADC and RD DTI parameters positively correlated with worsened histopathology at 12 months (Fig  7, bottom row). Beginning approximately 14 days to 1 month post TBI, ADC and RD values are significantly aberrantly elevated, however at 3 days after injury, we observed a clear reduction in these values. The observed correlation indicates that the less the initial decrease in these values, the greater the ipsilateral atrophy after moderate TBI was present. This unexpected correlation was not observed after severe TBI.

Discussion
In the current study, we evaluated how DTI and CBF PWI measurements evolved over time in a preclinical model of moderate and severe TBI, as well as the degree of chronic cerebral atrophy at 1 year after injury. Furthermore, we sought to establish whether there was a significant correlative relationship between MRI parameters and 12-month histopathology at various time points over the course of the study.
Consistent with the clinical literature, we detected DTI and PWI alterations that reflected injury-induced aberrations observed in TBI patients [57]. In all structures analyzed, we found

Fig 7. DTI and PWI modalities as prognostic indicators after moderate and severe TBI. (Top row)
At one month after severe TBI, there was a significant correlation between ADC and PWI in the ipsilateral cortex. Lower, more normalized ADC values correlated to greater CBF perfusion. Six months after severe injury, reduced CBF perfusion in the ipsilateral cortex significantly correlated with worsened degree of chronic cortical atrophy at 12 months. (Middle row) Correlation analyses showed that at 6 and 8 months after severe TBI, there was a positive correlation between FA values and PWI CBF. The larger the FA value, more closely resembling sham uninjured levels, the greater the degree of CBF perfusion in the ipsilateral hippocampus. (Bottom row) At an acute 3-day time point, ADC and RD were noticeably reduced in the ipsilateral corpus callosum of moderate TBI animals. We observed a significant positive correlation in which the greater the value of ADC and RD, the worse the extent of atrophic histopathology after moderate TBI. These findings illustrated that the degree of DTI reduction at 3 days after moderate injury was significantly correlative of 12-month cytoprotection. Statistical details for correlation analyses can be found in the Materials and Methods section. https://doi.org/10.1371/journal.pone.0289786.g007

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that ADC dropped at an acute 3-day time point, reaching significance between moderate and sham groups in both the hippocampus and external capsule. These ADC changes are consistent with experimental clinical findings in which axial diffusivity is reported to be significantly reduced at acute time points in patients, potentially related to persistent axonal injury, demyelination, and vasogenic and cytotoxic edema [33]. In terms of the injury model, previous studies have documented temporal and regionally specific structural changes in gray and white matter structures including BBB, axonal damage and visually edematous appearing tissue [40,42,51,58]. We further speculate that acute ADC decreases could be due to a reduction of the extracellular space secondary to injury-associated ischemic changes. Blood flow studies have shown that moderate and severe FPI produced ischemic levels in areas of the evolving contusion [41,43]. In this regard, Shen and colleagues reported in acute stroke patients a hyperacute reduction in ADC values that was attributed to the observed slowdown of water molecules by cellular edema post-infarction [35]. The mechanisms related to cytotoxic edema are often associated with ischemia-mediated disruption of metabolism and loss of ionic gradients, which can increase the flow of water from the extracellular space into intracellular compartments. Additional factors can also contribute to a reduction in water molecule diffusions, such as the subsequent decrease of extracellular space and increased viscosity [59]. Beginning at 14 days to 1 month, we observed that ADC began to increase within all structures analyzed after both moderate and severe TBI and remained elevated throughout the duration of the imaging. These findings are consistent with microstructural changes after TBI in which an increase in water diffusion is associated with loss of neuronal tissue, cavitation, and injury-induced gliosis [60].
In the present study, we found a significant reduction in FA values in the ipsilateral hippocampus, corpus callosum, and external capsule after moderate and severe TBI. The cortex demonstrated biphasic FA changes. FA reduction implies axonal pathology, and it has been reported that increased FA values after the injury is associated with better outcomes and reparative reorganization of axonal fibers [61]. Yuh et al. reported that severely reduced FA values after TBI in patients presenting with intracranial lesions were a significant predictor of poor 3and 6-month GOS-E outcome measures, and superior to conventional imaging and demographic/socioeconomic characteristics [62].
Radial diffusivity (RD) was the third DTI metric we assessed longitudinally after TBI. Our studies have documented patterns of axonal damage using β-APP [51]. We observed significant pathological elevations at a majority of imaging time points in the hippocampus and in major white matter tracts corpus callosum and external capsule. Our findings showed significantly increased RD in 3 of 4 structures across the majority of time points. Importantly, this pathological increase has been shown to be associated with the development and severity of post-concussive syndrome [63]. In another study, Perez and colleagues showed that increased RD values after traumatic axonal injury (TAI) associated brain injury was related to the degree of injury severity, while parallel diffusivity was an indicator of neuronal connectivity speed and axonal tract integrity. Furthermore, increased RD may be a contributing factor to reduced FA values [64].
The study used PWI to measure cerebral blood flow (CBF) between injured and noninjured ipsilateral cortices and hippocampi across time points. We observed decreased CBF at virtually all time points, reaching significance most frequently in chronic periods. We did not observe progressive changes in gray matter CBF after a 4-month time point, indicating that injury-induced blood flow reduction was sustained out to one year, the end point of the present study-which is consistent with published reports on patients with moderate to severe TBI [65]. When CBF is pathologically reduced, there is disruption of metabolic processes and a mismatch between supply and demand for oxygen and glucose, leading to cell death, long-term impairment, and dysfunction [47,[66][67][68][69]. There is a significant positive correlation between injury-induced lowered CBF and cognitive deficits, even in mild TBI [70,71]. Previous studies have shown that a reduction in CBF correlates with reduction in ADC, signifying ischemia-related changes [72], which, notably, is consistent with our findings at 1 month post severe TBI in the cortex.
In addition to reporting longitudinal DTI and PWI changes across 7 acute and chronic time points after moderate and severe TBI, we also analyzed the degree of cerebral atrophy at 1 year post TBI. As mentioned previously, there was highly significant asymmetry between ipsilateral and contralateral volumes in all structures evaluated at this chronic timepoint, which is consistent with the literature [58,73]. The degree of atrophy trended in an injury-severity dependent manner in all ROIs except for the hippocampus.
Given the copious amount of information provided using multiple MRI modalities, an important goal of this study was to determine whether there were significant correlations between measurements and the degree to which these modalities could be utilized as prognostic indicators of long-term outcomes. In the present study, we found that chronic DTI and PWI values correlated significantly after severe TBI in the cortex and hippocampus. Furthermore, we determined that the greater the aberrancy of ADC and FA measurements, the greater the pathological reductions in CBF. Thus, ADC and FA values could be used to predict the magnitude of diminished perfusion in the severely injured cortex and hippocampus, and vice versa.
We also found that, at a chronic 6-month time point, the extent of decreased CBF was indicative of the magnitude of cortical atrophy at 12 months. A direct and significant correlation (p-value = 0.001) between these two variables signified that a loss of CBF perfusion is suggestive of patterns of worsened histopathological outcomes [41]. Causality, however, for example whether decreases in CBF contributed to atrophic tissue progression, cannot be addressed in the current study.
As shown and discussed above, ADC and RD measurements are significantly and pathologically elevated after TBI across time points, except in the acute 3-day phase in which these metrics drop below sham levels. Interestingly, we found that the degree of this drop in ADC and RD values measured in the corpus callosum may be indicative of some endogenous protective mechanism that results in lessened histopathology at 12 months. Further investigation of contributing factors is warranted.
In the present study assessing MRI modalities in moderate and severe FPI, we did not observe extensive significance between injury severity groups. At 8 months, we found that in all three DTI metrics assessed, severe TBI animals had greater diffusion abnormalities in major white matter tracts relative to moderately injured animals. In our preclinical fluid-percussion TBI model, a moderate injury pulse ranges between 1.8 and 2.2 ATM while severe TBI is defined between by 2.3 to 2.5 [41,51]. In the present study, the average moderate injury ATM was 1.99 and severe animals averaged 2.3 ATM. We speculate that perhaps the pressure pulse was not disparate enough to cause multiple significant MRI alterations between injury groups, except at an 8-month time point. Why injury severity differences were only evident at this specific posttraumatic time point remains to be determined. Given the progressive nature of TBI, it is possible that this chronic time point captured the peak of diffusion abnormalities in major white matter tracts, and by 12 months, endogenous protective mechanisms are sufficiently robust enough to resolve a portion of the remaining white matter dysfunction in severely injured animals. It would be valuable to assess MRI metrics of differing degrees of injury severity in additional models of TBI, such as in closed cortical impact or in concussive weight drop TBI models. An additional consideration for this study would be to increase the number of animals per group although our sample size analysis indicated that 8-10 animals per groups would be sufficient for detecting a significant difference.
There are inherent limitations to using DTI for evaluating comparative changes in microstructural anisotropy. Averaged DTI voxels on the scale of MRI measurements have limited precision in differentiating changes in white matter fiber orientation, especially in longerrange or highly complex tracts [74]. Furthermore, there is much heterogenicity when assessing DTI metrics in clinical populations, and thus it can be challenging to draw conclusions based on DTI values alone. That being said, Hulkower et al., in a comprehensive review, showed that aberrant reductions in FA values, which are the most frequently reported DTI metric across studies, were positively correlated to attention, executive function, and memory impairment [75]. However, conflicting reports are not uncommon, underscoring the widespread and inherent heterogeneity of brain injury and DTI measurements [76]. Given the varying degrees of diffusion abnormalities we observed across ADC, FA, and RD in all ROIs (i.e. in the ipsilateral hippocampus, FA was only significant at 8 and 12 months while ADC values were significantly different at every assessed time point), we emphasize the importance of using multiple DTI metrics to achieve a more accurate and precise understanding of the extent of the injuryinduced disruption of white matter integrity.
Female rats were not utilized in the present study as hormonal fluctuations with the female reproductive cycle can introduce biological variability. Whether MRI patterns, histological findings, and correlative outcomes are applicable to TBI in female rats remain to be determined as gender is a significant biological variable. Furthermore, repeated exposure to gaseous anesthetic, such as what was conducted at each MRI session, could potentially cause unanticipated changes unrelated to TBI. However, at levels administered, we do not anticipate significant effects on outcomes.
In this study, we demonstrated that non-invasive MRI provided valuable information for identifying microstructural abnormalities in the brain after TBI. Utilizing longitudinal imaging out to one year after moderate and severe injury allowed for the visualization of how DTI and PWI metrics vary over time. We also identified statistically significant correlations among MRI modalities as well as to chronic histopathological atrophy as a result of the progressive nature of the injury in both moderate and severe TBI.
The temporal and regional changes using a multimodal analytical approach offers a powerful strategy to identify therapeutic targets. Recognizing these alterations and identifying various windows for intervention allows for the potential of MRI modalities as essential long-term predictors of TBI-induced sequelae and for implementation of time-targeted treatment therapies in the clinical setting.
Supporting information S1